Process for the synthesis of oligomeric compounds

ABSTRACT

Synthetic processes are provided wherein oligomeric compounds are prepared having phosphodiester, phosphorothioate, phosphorodithioate, or other covalent linkages. Also provided are synthetic intermediates useful in such processes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/349,659filed Jul. 8, 1999 which is a continuation-in-part U.S. application Ser.No. 09/111,678 filed Jul. 8, 1998 now U.S. Pat. No 6,326,478.

FIELD OF THE INVENTION

This invention relates to methods for the preparation of oligomericcompounds having phosphite, phosphodiester, phosphorothioate,phosphorodithioate or other linkages, and to intermediates useful intheir preparation.

BACKGROUND OF THE INVENTION

Oligonucleotides and their analogs have been developed and used inmolecular biology in a variety of procedures as probes, primers,linkers, adapters, and gene fragments. Modifications to oligonucleotidesused in these procedures include labeling with nonisotopic labels, e.g.fluorescein, biotin, digoxigenin, alkaline phosphatase, or otherreporter molecules. Other modifications have been made to the ribosephosphate backbone to increase the nuclease stability of the resultinganalog. Examples of such modifications include incorporation of methylphosphonate, phosphorothioate, or phosphorodithioate linkages, and2′-O-methyl ribose sugar units. Further modifications include those madeto modulate uptake and cellular distribution. With the success of thesecompounds for both diagnostic and therapeutic uses, there exists anongoing demand for improved oligonucleotides and their analogs.

It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic or otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. For disease states, classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease-causing or disease-potentiatingfunctions. In newer therapeutic approaches, modulation of the actualproduction of such proteins is desired. By interfering with theproduction of proteins, the maximum therapeutic effect may be obtainedwith minimal side effects. It is therefore a general object of suchtherapeutic approaches to interfere with or otherwise modulate geneexpression, which would lead to undesired protein formation.

One method for inhibiting specific gene expression is with the use ofoligonucleotides, especially oligonucleotides which are complementary toa specific target messenger RNA (mRNA) sequence. Severaloligonucleotides are currently undergoing clinical trials for such use.Phosphorothioate oligonucleotides are presently being used as suchantisense agents in human clinical trials for various disease states,including use as antiviral agents.

Transcription factors interact with double-stranded DNA duringregulation of transcription. Oligonucleotides can serve as competitiveinhibitors of transcription factors to modulate their action. Severalrecent reports describe such interactions (see Bielinska, A., et. al.,Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89,203-209).

In addition to such use as both indirect and direct regulators ofproteins, oligonucleotides and their analogs also have found use indiagnostic tests. Such diagnostic tests can be performed usingbiological fluids, tissues, intact cells or isolated cellularcomponents. As with gene expression inhibition, diagnostic applicationsutilize the ability of oligonucleotides and their analogs to hybridizewith a complementary strand of nucleic acid. Hybridization is thesequence specific hydrogen bonding of oligomeric compounds viaWatson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases ofsuch base pairs are said to be complementary to one another.

Oligonucleotides and their analogs are also widely used as researchreagents. They are useful for understanding the function of many otherbiological molecules as well as in the preparation of other biologicalmolecules. For example, the use of oligonucleotides and their analogs asprimers in PCR reactions has given rise to an expanding commercialindustry. PCR has become a mainstay of commercial and researchlaboratories, and applications of PCR have multiplied. For example, PCRtechnology now finds use in the fields of forensics, paleontology,evolutionary studies and genetic counseling. Commercialization has ledto the development of kits which assist non-molecular biology-trainedpersonnel in applying PCR. Oligonucleotides and their analogs, bothnatural and synthetic, are employed as primers in such PCR technology.

Oligonucleotides and their analogs are also used in other laboratoryprocedures. Several of these uses are described in common laboratorymanuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J.Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; andCurrent Protocols In Molecular Biology, F. M. Ausubel, et al., Eds.,Current Publications, 1993. Such uses include as syntheticoligonucleotide probes, in screening expression libraries withantibodies and oligomeric compounds, DNA sequencing, in vitroamplification of DNA by the polymerase chain reaction, and insite-directed mutagenesis of cloned DNA. See Book 2 of MolecularCloning, A Laboratory Manual, supra. See also “DNA-protein interactionsand The Polymerase Chain Reaction” in Vol. 2 of Current Protocols InMolecular Biology, supra.

Oligonucleotides and their analogs can be synthesized to have customizedproperties that can be tailored for desired uses. Thus a number ofchemical modifications have been introduced into oligomeric compounds toincrease their usefulness in diagnostics, as research reagents and astherapeutic entities. Such modifications include those designed toincrease binding to a target strand (i.e. increase their meltingtemperatures, Tm), to assist in identification of the oligonucleotide oran oligonucleotide-target complex, to increase cell penetration, tostabilize against nucleases and other enzymes that degrade or interferewith the structure or activity of the oligonucleotides and theiranalogs, to provide a mode of disruption (terminating event) oncesequence-specifically bound to a target, and to improve thepharmacokinetic properties of the oligonucleotide.

The chemical literature discloses numerous processes for couplingnucleosides through phosphorous-containing covalent linkages to produceoligonucleotides of defined sequence. One of the most popular processesis the phosphoramidite technique (see, e.g., Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.;Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references citedtherein), wherein a nucleoside or oligonucleotide having a free hydroxylgroup is reacted with a protected cyanoethyl phosphoramidite monomer inthe presence of a weak acid to form a phosphite-linked structure.Oxidation of the phosphite linkage followed by hydrolysis of thecyanoethyl group yields the desired phosphodiester or phosphorothioatelinkage.

The phosphoramidite technique, however, has significant disadvantages.For example, cyanoethyl phosphoramidite monomers are quite expensive.Although considerable quantities of monomer go unreacted in a typicalphosphoramidite coupling, unreacted monomer can be recovered, if at all,only with great difficulty.

Another disadvantage of using a β-eliminating cyanoethoxy group isformation of acrylonitrile upon removal of the phosphorus protectinggroup. Acrylonitrile is a highly toxic agent as well as a suspectedcarcinogen (See 1994-1995 Aldrich Chemical Company Catalog, at page 32).Acrylonitrile is also suspected of forming cyclic structures withthymidine resulting in oligomeric compounds having decreasedhybridization ability. These modified oligomeric compounds areundesirable because they are difficult to separate from the desiredoligomeric compound.

Consequently, there remains a need in the art for synthetic methods thatwill overcome these problems.

Several processes are known for the solid phase synthesis ofoligonucleotide compounds. These are generally disclosed in thefollowing U.S. Patents: U.S. Pat. No. 4,458,066; issued Jul. 3, 1984;U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No.5,132,418, issued Jul. 21, 1992. Additionally, a process for thepreparation of oligonucleotides using phosphoramidite intermediates isdisclosed in U.S. Pat. No. 4,973,679, issued Nov. 27, 1990.

A process for the preparation of phosphoramidites is disclosed in U.S.Pat. No. 4,415,732, issued Nov. 15, 1983.

Phosphoramidite nucleoside compounds are disclosed in U.S. Pat. No.4,668,777, issued May 26, 1987.

A process for the preparation of oligonucleotides using a β-eliminatingphosphorus protecting group is disclosed in U.S. Pat. No. Re. 34,069,issued Sep. 15, 1992.

A process for the preparation of oligonucleotides using a β-eliminatingor allylic phosphorus protecting group is disclosed in U.S. Pat. No.5,026,838, issued Jun. 25, 1991.

SUMMARY OF THE INVENTION

In one aspect of the present invention, methods are provided for thepreparation of oligomeric compounds comprising a moiety having theFormula I:

wherein:

A is a monocyclic or bicyclic aromatic ring system;

R¹¹ and R₁₂ are each independently H, alkyl, aryl, heteroalkyl,heteroaryl, alkaryl, or aralkyl;

or R₁₁ and R₁₂ together with the carbon atoms to which they are attachedform an optionally substituted aliphatic or aromatic ring having from 4to 6 ring atoms;

X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl,substituted sulfonyl, or substituted thio, wherein said substituent isalkyl, aryl, or alkaryl;

or X₄ is a group of formula —C (═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups;

or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

X₁ and X₅ are each independently O or S; comprising:

(a) providing a compound having the Formula II:

wherein:

each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl,C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro,nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino,hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule,conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a groupthat enhances the pharmacodynamic properties of oligonucleotides, or agroup that enhances the pharmacokinetic properties of oligonucleotides;

v is from 0 to about 10;

or R₁ has the formula:

y1 is 0 or 1;

y2 is independently 0 to 10;

y3 is 1 to 10;

E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substituted C₁-C₁₀alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered oruntethered conjugate group, a linker to a solid support; or Q₁ and Q₂,together, are joined in a nitrogen protecting group or a ring structurethat can include at least one additional heteroatom selected from N andO;

or R₁ has one of formula XI or XII:

 wherein

Z₀ is O, S, or NH;

q¹ is from 0 to 10;

q² is from 1 to 10;

q³ is 0 or 1;

q⁴ is, 0, 1 or 2;

Z₄ is OM₁, SM₁, or N(M₁)₂;

each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

M₂ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo,SQ₁ or CN;

R₃ is hydrogen, a hydroxyl protecting group, or a linker connected to asolid support;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

n is 0 to about 50;

M is an optionally protected internucleoside linkage;

R₅ is —N (R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen; and

R₆ is straight or branched chain alkyl having from 1 to 10 carbons; and

(b) reacting the compound of Formula II with a compound having FormulaIII:

wherein m is 0 to about 50;

R_(3a) is hydrogen;

R₂ is hydrogen, a hydroxyl protecting group, or a linker connected to asolid support, provided that R₂ and R_(3a) are not both simultaneously alinker connected to a solid support; to form the oligomeric compound.

Some preferred embodiments of the methods of the invention furthercomprise the step of oxidizing or sulfurizing the oligomeric compound.In some preferred embodiments, the methods of the invention furthercomprise transforming the oxidized or sulfurized oligomeric compound toform a further compound having the Formula III, where m is increasedby 1. Other prefered embodiments further comprise a capping step,performed prior to or subsequent to oxidation or sulfurization.

In some preferred embodiments, the methods of the invention furthercomprising the step of cleaving the oligomeric compound from the solidsupport to produce a compound having the Formula IV:

In some preferred embodiments of the methods of the invention, A isphenyl or a naphthalene.

In further preferred embodiments of the methods of the invention, X₄ isbenzoyl, acetyl (—C(═O)—CH₃) or levulinyl.

In some partiularly preferred embodiments, X₄ is benzoyl, acetyl orlevulinyl, A is phenyl, with the moiety —OX₄ being in the ortho or paraposition thereof, with the ortho position being more preferred.

In further preferred embodiments of the methods of the invention, X₄ isbenzoyl, acetyl or levulinyl, A is a naphthalene ring connected to X₅ atthe 1-position, with the moiety —OX₄ being in the 5- or 6-position ofthe naphthalene ring.

In further preferred embodiments of the invention, each R₆ is isopropyl.

In some especially preferred embodiments of the invention, n is 0. Infurther prefered embodiments, at least one of X₁ and X₅ is O. Morepreferably, X₁ and X₅ are each O.

In some especially preferred emboiments, n is 0; X₄ is benzoyl, acetylor levulinyl; A is phenyl; —OX₄ is in the ortho or para position, withthe ortho position being more preferred; X₁ and X₅ are each O; and R₅ isdiisopropylamino.

In some preferred embodiments, the compound of Formula II is obtained byreaction of a compound having Formula V:

with a compound having the Formula VI:

in the presence of an acid, preferably tetrazole. Preferably, R₅ isN,N-diisopropylamino.

In other preferred embodiments, the compound of Formula II is obtainedby (a) reacting a compound having Formula-V with a chlorophosphinecompund of formula ClP(R₅)₂ in the presence of a base; and

(b) contacting the product of step (a) with a compound of Formula XX:

in the presence of an acid. Preferably, the chlorophosphine compound hasthe formula ClP[(i-Pr)₂N]₂.

Also provided in accordance with the present invention are compoundshaving Formula VII:

wherein:

A is a monocyclic or bicyclic aromatic ring system;

R₁₁ and R₁₂ are each independently H, alkyl, aryl, heteroaryl, alkaryl,or aralkyl;

or R₁₁ and R₁₂ together with the carbon atoms to which they are attachedform an optionally substituted aliphatic or aromatic ring having from 4to 6 ring atoms;

X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl,substituted sulfonyl, or substituted thio, wherein said substituent isalkyl, aryl, or alkaryl;

or X₄ is a group of formula —C (═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups;

or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

X₁ and X₅ are each independently O or S;

D is (R₇) (R₈) P—or (R₇) (R₈) P (═X₂)—;

R₈ is R₅, or has the Formula VIII:

wherein:

each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl,C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro,nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino,hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule,conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a groupthat enhances the pharmacodynamic properties of oligonucleotides, or agroup that enhances the pharmacokinetic properties of oligonucleotides;

v is from 0 to about 10;

or R₁ has the formula:

y1 is 0 or 1;

y2 is independently 0 to 10;

y3 is 1 to 10;

E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substituted C₁-C₁₀alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered oruntethered conjugate group, a linker to a solid support; or Q₁ and Q₂,together, are joined in a nitrogen protecting group or a ring structurethat can include at least one additional heteroatom selected from N andO;

or R₁ has one of formula XI or XII:

 wherein

Z⁰ is O, S, or NH;

q¹ is from 0 to 10;

q² is from 1 to 10;

q³ is 0 or 1;

q⁴ is, 0, 1 or 2;

Z₄ is OM₁, SM₁, or N(M₁) ₂;

each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

M₂ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo,SQ₁ or CN;

each X₂ is O or S;

R₅ is —N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen;

M is an optionally protected internucleoside linkage;

m is 0 to about 50;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase; and

R₇ is R₅, or has the Formula IX:

wherein:

R₃ is hydrogen, a hydroxyl protecting group, or a linker connected to asolid support;

n is 0 to about 50; with the proviso that the sum of m and n do notexceed 50.

In some preferred embodiments, X₄ is benzoyl, acetyl or levulinyl, withacetyl being preferred.

In some partiularly preferred embodiments, X₄ is benzoyl, acetyl orlevulinyl, A is phenyl, with the moiety —OX₄ being in the ortho or paraposition, with the ortho position being more preferred, and R₁₁ and R₁₂are each H.

In further preferred embodiments of the methods of the invention, X₄ isbenzoyl, acetyl or levulinyl, A is a naphthalene ring connected to X₅ atthe 1-position, with the moiety —OX₄ being at the 5- or 6-position ofthe naphthalene ring, and R₁₁ and R₁₂ are each H.

In some preferred embodiments, at least one of X₁ and X₅ is O. In morepreferred embodiments, X₁ and X₅ are each O.

In particularly preferred embodiments, X₄ is benzoyl, acetyl orlevulinyl, A is phenyl with —OX₄ being in the ortho or para position, X₁and X₅ are each O, and R₁₁ and R₁₂ are each H.

In some preferred embodiments, R₈ is R₅. In further preferredembodiments, n is 0. In still further preferred embodiments, R₈ is R₅, nis 0, X₄ is benzoyl, acetyl or levulinyl, A is phenyl with —OX₄ attachedat the ortho or para position; X₁ and X₅ are each O, and R₁₁ and R₁₂ areeach H.

In some preferred embodiments, R₈ has the Formula VIII, and R₇ has theFormula IX. In further preferred embodiments, R₈ has the Formula VIII,and R₇ has the Formula IX, and n is 0. In still further preferredembodiments, R₈ has the Formula VIII, and R₇ has the Formula IX, and nis 0 and m is 0. In still further preferred embodiments, R₈ has theFormula VIII, and R₇ has the Formula IX, n is 0, X₄ is benzoyl, acetylor levulinyl, A is phenyl with —OX₄ attached at the ortho or paraposition, X₁ and X₅ are each O, R₅ is diisopropylamino, and R₁₁ and R₁₂are each H.

In some preferred embodiments, at least one of X₁ and X₅ is S. Infurther preferred embodiments, A is (R₇) (R₈)P—.

In some preferred embodiments, the present invention provides compoundscomprising a moiety of Formula:

wherein the constituent variables are as previously defined.

Preferably, the moiety A is phenyl with —OX₄ attached at the ortho orpara position, with the ortho position being preferred; or A isnaphthalene connected to X₅ at the 1-position, and the moiety —OX₄ isattached to the 5- or 6-position of the naphthalene ring. In especiallypreferred embodiments, X₄ is benzoyl, acetyl or levulinyl, A is phenylwith —OX₄ is in the ortho or para position, and X₁ and X₅ are each O.

The present invention also provides compounds having Formula X:

or Formula XI:

wherein m and n are each independently from 0 to about 50, provided thatthe sum of m and n does not exceed 50; and the other constituentvariables are as previously defined.

In some preferred embodiments, R₂ is a linker connected to a solidsupport.

Also provided in accordance with the present invention are methods forthe preparation of a compound of Formula II:

wherein the consitituent variables are as previously defined,comprising:

(a) selecting a 5′-protected nucleoside having Formula V:

and

(b) reacting the nucleoside with a compound having the Formula VI:

in the presence of an acid.

The present invention also provides methods for the preparation of acompound of Formula II comprising:

(a) selecting a 5′-protected nucleoside of Formula V:

(b) reacting the protected nucleoside with a chlorophosphine compund offormula ClP(R₅)₂ in the presence of a base; and

(c) contacting the product of step (b) with a compound of Formula XX:

in the presence of an acid;

to form the nucleoside phosphoramidite.

In some preferred embodiments of the compounds of Formulas X and XI mand n are each 0.

Also provided in accordance with the present invention are compoundshaving the formula:

wherein A, X₁, X₄ X₅, R₁₁ and R₁₂ are as defined above, X₂ is halogen,and X₃ is —N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen. In further preferred embodiments, Ais phenyl with —O—X₄ in the ortho or para position, X₁ and X₅ are O, andR₁₁ and R₁₂ are each H, and X₄ is benzoyl, acetyl or levulinyl. In stillfurther preferred embodiments, X₃ is —N(R₆) ₂ where R₆ is isopropyl.Preferably, X₂ is chlorine.

The present invention also provides products produced by the methods ofthe invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides methods for the preparation of oligomericcompounds having phosphite, phosphodiester, phosphorothioate, orphosphorodithioate linkages, and to intermediates useful in theirpreparation.

In some preferred embodiments of the invention, methods are provided forthe preparation of an oligomeric compound comprising at least one moietyhaving the Formula I:

wherein:

A is a monocyclic or bicyclic aromatic ring system;

R₁₁ and R₁₂ are each independently H, alkyl, aryl, heteroaryl, alkaryl,or aralkyl;

or R₁₁ and R₁₂ together with the carbon atoms to which they are attachedform an optionally substituted aliphatic or aromatic ring having from 4to 6 ring atoms;

X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl,substituted sulfonyl, or substituted thio, wherein said substituent isalkyl, aryl, or alkaryl;

or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups;

or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

X₁ and X₅ are each independently O or S; comprising:

(a) providing a compound having the Formula II:

wherein:

each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl,C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro,nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkyl,NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino,hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule,conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a groupthat enhances the pharmaco-dynamic properties of oligonucleotides, or agroup that enhances the pharmacokinetic properties of oligonucleotides;

v is from 0 to about 10;

or R₁ has the formula:

y1 is 0 or 1;

y2 is independently 0 to 10;

y3 is 1 to 10;

E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substituted C₁-C₁₀alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered oruntethered conjugate group, a linker to a solid support; or Q₁ and Q₂,together, are joined in a nitrogen protecting group or a ring structurethat can include at least one additional heteroatom selected from N andO;

or R₁ has one of formula XI or XIII:

 wherein

Z₀ is O, S, or NH;

q₁ is from 0 to 10;

q² is from 1 to 10;

q³ is 0 or 1;

q⁴ is, 0, 1 or 2;

Z₄ is OM₁, SM₁, or N(M₁)₂;

each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

M₂ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo,SQ₁ or CN;

R₃ is hydrogen, a hydroxyl protecting group, or a linker connected to asolid support;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

n is 0 to about 50;

M is an optionally protected internucleoside linkage;

R₅ is —N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen; and

R₆ is straight or branched chain alkyl having from 1 to 10 carbons; and

(b) reacting the compound of Formula II with a compound having FormulaIII:

 wherein m is 0 to about 50;

R_(3a) is hydrogen;

R₂ is hydrogen, a hydroxyl protecting group, or a linker connected to asolid support, provided that R₂ and R_(3a) are not both simultaneously alinker connected to a solid support;

to form the oligomeric compound.

The methods of the present invention are useful for the preparation ofoligomeric compounds containing monomeric subunits that are joined by avariety of linkages, including phosphite, phosphodiester,phosphorothioate, and/or phosphorodithioate linkages. As used herein,the term “oligomeric compound” is used to refer to compounds containinga plurality of nucleoside monomer subunits that are joined byinternucleoside linkages, preferably phosphorus-containing linkages,such as phosphite, phosphodiester, phosphorothioate, and/orphosphorodithioate linkages. The term “oigomeric compound” thereforeincludes naturally occurring oligonucleotides, their analogs, andsynthetic oligonucleotides. Monomer or higher order synthons havingFormulas II or III above include both native (i.e., naturally occurring)and synthetic (e.g., modified native or totally synthetic) nucleosidesand nucleotides.

In some preferred embodiments, a phosphoramidite protected at the5′-position is reacted with the 3′-hydroxyl group of a compound ofFormula III to produce phosphite compound containing the linkage ofFormula I. Preferably, capping and/or oxidation or sulfurization stepsare then performed to produce a compound of Formula IV.

Methods for coupling compounds of Formula II and Formula III of theinvention include both solution phase and solid phase chemistries.Representative solution phase techniques are described in U.S. Pat. No.5,210,264, which is assigned to the assignee of the present invention.In preferred embodiments, the methods of the present invention areemployed for use in iterative solid phase oligonucleotide syntheticregimes. Representative solid phase techniques are those typicallyemployed for DNA and RNA synthesis utilizing standard phosphoramiditechemistry, (see, e.g., Protocols For Oligonucleotides And Analogs,Agrawal, S., ed., Humana Press, Totowa, N.J., 1993, hereby incorporatedby reference in its entirety). A preferred synthetic solid phasesynthesis utilizes phosphoramidites as activated phosphate compounds. Inthis technique, a phosphoramidite monomer is reacted with a freehydroxyl on the growing oligomer chain to produce an intermediatephosphite compound, which is subsequently oxidized to the p^(v) stateusing standard methods. This technique is commonly used for thesynthesis of several types of linkages including phosphodiester,phosphorothicate, and phosphorodithioate linkages.

Typically, the first step in such a process is attachment of a firstmonomer or higher order subunit containing a protected 5′-hydroxyl to asolid support, usually through a linker, using standard methods andprocedures known in the art. See for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,hereby incorporated by reference in its entirety. The support-boundmonomer or higher order first synthon is then treated to remove the5′-protecting group, to form a compound of Formula III wherein R₂ is alinker connected to a solid support. Typically, this is accomplished bytreatment with acid. The solid support bound monomer is then reactedwith a compound of Formula II to form a compound of Formula IV, whichhas a phosphite or thiophosphite linkage of Formula I. In preferredembodiments, synthons of Formula II and Formula III are reacted underanhydrous conditions in the presence of an activating agent such as, forexample, 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, ordiisopropylamino tetrazolide.

In some preferred embodiments, phosphite or thiophosphite compoundscontaining a linkage of Formula I are oxidized or sulfurized as shownbelow to produce compounds having a linkage of Formula XII, where X₁ andX₂ can each be O or S:

Choice of oxidizing or sulfurizing agent will determine whether thelinkage of Formula I will be oxidized or sulfurized to aphosphotriester, thiophosphotriester, or a dithiophosphotriesterlinkage.

It is generally preferable to perform a capping step, either prior to orafter oxidation or sulfurization of the phosphite triester,thiophosphite triester, or dithiophosphite triester. Such a capping stepis generally known to be beneficial by preventing shortened oligomerchains, by blocking chains that have not reacted in the coupling cycle.One representative reagent used for capping is acetic anhydride. Othersuitable capping reagents and methodologies can be found in U.S. Pat.No. 4,816,571, issued Mar. 28, 1989, hereby incorporated by reference inits entirety.

Treatment with an acid removes the 5′-hydroxyl protecting group, andthus transforms the solid support bound oligomer into a further compoundof Formula III wherein R_(3a) is hydrogen, which can then participate inthe next synthetic iteration; i.e., which can then be reacted with afurther compound of Formula II. This process is repeated until anoligomer of desired length is produced.

The completed oligomer is then cleaved from the solid support. Thecleavage step, which can precede or follow deprotection of protectedfunctional groups, will in prefered embodiments yield a compound havingFormula IV wherein R₂ is hydrogen. During cleavage, the linkages betweenmonomeric subunits are converted from phosphotriester,thiophosphotriester, or dithiophosphotriester linkages tophosphodiester, phosphorothioate, or phosphorodithioate linkages.

Without intending that the invention be bound by any particular theory,it is believed that the loss of the oxygen or sulfur protecting groupwhere X₄ is an alkanoyl (e.g., acetyl) group occurs via a fragmentationmechanism, illustrated in Scheme I below for embodiments wherein moietyA is phenyl with the group —OX₄ (exemplified as an acetyl group) in thepara position:

In this mechanism, a nucleophile (for example, ammonia) first attacksthe carbonyl carbon of the acetoyl group. The resonant movement ofelectrons as depicted in Scheme I above is believed to cause the loss ofthe oxygen or sulfur protecting group via a fragmentation, therebyforming a phosphodiester, phosphorothioate, or phosphorodithioatelinkage. The other products of the deprotection are Nu—C(═O)—CH₃,p-quinone, and ethylene gas.

The mechanism for embodiments wherein moiety A is phenyl with the group—OX₄ (exemplified as an acetyl group) attached to the ortho position isshown below in Scheme II:

The products of the deprotection are the unprotected linkage,Nu-C(═O)—CH₃, o-quinone, and ethylene gas.

In some preferred embodiments of the compounds of the invention,substituent X₄ is selected such that it facilitates attack by anucleophile, or a base. Accordingly, X₄ can be any of a variety ofsubstituents, provided that it does not otherwise interfere with themethods of the invention. Preferred non-silyl X₄ groups include alkarylgroups, sulfoxyl groups, sulfonyl groups, thio groups, substitutedsulfoxyl groups, substituted sulfonyl groups, or substituted thiogroups, wherein the substituents are selected from the group consistingof alkyl, aryl, or alkaryl. More preferred non-silyl X₄ groups includecompounds of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 and R₄₀ islower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl,aralkyl or heteroaryl groups are optionally substituted with one or morealkyl, aryl, aralkyl, halo or acetyl groups. Particularly preferred X₄groups include acetyl (—C(═O)—CH₃), benzoyl (—C(═O)—Ph), phenylacetyl(—C(═O)—CH₂—Ph) and levulinyl (—C(═O)—(CH₂)₂—C(═O) —CH₃) groups.

In one embodiment of the invention the moiety —OX₄ forms a carbonate orsubstituted carbonate group. In some preferred embodiments, X₄ has theformula —C(═O)—(O)_(aa)—R₄₀ where aa is 1 and R₄₀ is lower alkyl, aryl,aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl orheteroaryl groups are optionally substituted with one or more alkyl,aryl, aralkyl, halo or acetyl groups. Carbonate protecting groups arediscussed in for example, Green and Wuts, Protective Groups in OrganicSynthesis, 2d edition, John Wiley & Sons, New York, 1991, pages 104-105et al., incorporated herein by reference.

X₄ can also be a group of formula a group of formula—(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1, and each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms. While not wishing to be bound by a particulartheory, it is believed that the loss of the oxygen or sulfur where X₄ isa trisubstituted silyl moiety, occurs via a fragmentation mechanism,illustrated in Scheme III below for embodiments wherein A is phenyl with—OX₄ at the para position:

In this mechanism, a nucleophile attacks the silyl silicom atom, and theresonant movement of electrons as depicted in Scheme III above isbelieved to cause the loss of the oxygen or sulfur protecting group viaa fragmentation mechanism, thereby forming a phosphodiester,phosphorothioate, or phosphorodithioate linkage. The other products ofthe deprotection are believed to be ethylene gas, p-quinone, and acompound of formula Nu—Si(R₉)₃. For embodiments wherein the moiety(R₉)₃Si— is in the ortho position of the phenyl ring, the analogousfragmentation is beleived to result in the same products, except for theproduction of o-quinone instead of p-quinone. For embodiments wherein dis 1, it is believed that a similar fragmentation mechanism wouldproduce the same products, and one additional mole of ethylene.

A wide variety of bases or nucleophiles can be used to initiate thefragmentation of the oxygen or sulfur protecting groups describedherein. These include ammonium hydroxide, fluoride ion, alkyl amines,aqueous bases, and alkyl amines in combination with ammonium hydroxide.The resulting products include phosphate, phosphorothioate, andphosphorodithioate containing compounds.

Contact with fluoride ion preferably is effected in a solvent such astetrahydrofuran, acetonitrile, dimethoxyethane, or water. Fluoride ionpreferably is provided in the form of one or more salts selected fromtetraalkylammonium fluorides (e.g., tetrabutylammonium fluoride (TBAF)),potassium fluoride, or cesium fluoride.

Preferably, conditions for removal of the oxygen or sulfur protectinggroup via fragmentation mechanisms described above also effect cleavageof the oligomeric compound from the solid support.

The methods of the present invention are useful for the preparation ofoligomeric compounds from monomeric or oligomeric amidite synthons, forexample synthons having Formula II. The internucleoside linkages of sucholigomeric amidite synthons, represented by moiety M in the compoundsand methods described herein, can be any internucleoside linkage as isknown in the art, including phosphorus based linking groups such asphosphite, phosphodiester, phosphorothioate, and phosphorodithioatelinkages, and other linkages known in the art. Such linkages can beprotected, i.e., they can bear, for example, phosphate protectinggroups. Included with the definition of internucleoside linkages aregroups described herein, having the Formula:

In preferred embodiments, the methods of the invention are used for thepreparation of oligomeric compounds including oligonucleotides and theiranalogs. As used herein, the term “oligonuclotide analog” meanscompounds that can contain both naturally occurring (i.e. “natural”) andnon-naturally occurring (“synthetic”) moieties, for example, nucleosidicsubunits containing modified sugar and/or nucleobase portions. Sucholigonucleotide analogs are typically structurally distinguishable from,yet functionally interchangeable with, naturally occurring or syntheticwild type oligonucleotides. Thus, oligonucleotide analogs include allsuch structures which function effectively to mimic the structure and/orfunction of a desired RNA or DNA strand, for example, by hybridizing toa target. The term synthetic nucleoside, for the purpose of the presentinvention, refers to a modified nucleoside. Representative modificationsinclude modification of a heterocyclic base portion of a nucleoside togive a non-naturally occurring nucleobase, a sugar portion of anucleoside, or both simultaneously.

Representative nucleobases useful in the compounds and methods describedherein include adenine, guanine, cytosine, uridine, and thymine, as wellas other non-naturally occurring and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo,oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adeninesand guanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine. Further naturally and non naturallyoccurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808(Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, P. D., Anti-Cancer DrugDesign, 1991, 6, 585-607, each of which are hereby incorporated byreference in their entirety. The term ‘nucleosidic base’ is furtherintended to include heterocyclic compounds that can serve as likenucleosidic bases including certain ‘universal bases’ that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as a universal base is 3-nitropyrrole.

As used herein the term “2′-substituent group” denotes groups attachedto the 2′ position of the ribosyl moiety, with or without an oxygenatom.

Preferred 2′-substituent groups described herein are represented in thecompounds described herein by the variable R₁, which can beindependently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl, C₂-C₂₀alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro,nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino,hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule,conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a groupthat enhances the pharmacodynamic properties of oligonucleotides, or agroup that enhances the pharmacokinetic properties of oligonucleotides;

v is from 0 to about 10;

or R₁ has the formula:

y1 is 0 or 1;

y2 is independently 0 to 10;

y3 is 1 to 10;

E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substituted alkyl,dialkylaminoalkyl, a nitrogen protecting group, a tethered or untetheredconjugate group, a linker to a solid support; or Q₁ and Q₂, together,are joined in a nitrogen protecting group or a ring structure that caninclude at least one additional heteroatom selected from N and O;

or R₁ has one of formula XI or XII:

 wherein

Z₀ is O, S, or NH;

q₁ is from 0 to 10;

q² is from 1 to 10;

q³ is 0 or 1;

q⁴ is, 0, 1 or 2;

Z₄ is OM₁, SM₁, or N(M₁)₂;

each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

M₂ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo,SQ₁ or CN.

Representative 2′—O—sugar substituents of formula XI are disclosed inU.S. patent application Ser. No.: 09/130,973, filed Aug. 7, 1998,entitled Capped 2′-Oxyethoxy Oligonucleotides, hereby incorporated byreference in its entirety.

Representative cyclic 2′—O—sugar substituents of formula XII aredisclosed in U.S. patent application Ser. No.: 09/123,108, filed Jul.27, 1998, entitled RNA Targeted 2′-Modified Oligonucleotides that areConformationally Preorganized, hereby incorporated by reference in itsentirety.

One particularly preferred group includes 2′-methoxyethoxy[2′—O—CH₂CH₂OCH₃, also known as 2′—O—(2-methoxyethyl) or 2′-MOE] (Martinet al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. Afurther referred modification includes 2′-dimethylaminooxyethoxy, i.e.,a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inco-owned U.S. patent application Ser. No. 09/016,520, filed on Jan. 30,1998, the contents of which are herein incorporated by reference. Otherpreferred modifications include 2′-methoxy (2′—O—CH₃) and2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂).

Further preferred 2′-sugar modifications amenable to the presentinvention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, andpolyethers of the formula (O-alkyl)_(m) where m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi, et al., Drug Design and Discovery1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardoet. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9,249, each of which are hereby incorporated by reference in theirentirety. Further sugar modifications are disclosed in Cook, P. D.,Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O-alkyl,O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl aminosubstitution is described in U.S. patent application Ser. No.08/398,901, filed Mar. 6, 1995, entitled Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions, herebyincorporated by reference in its entirety.

Additional 2′ sugar modifications amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, where each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR nucleosides are disclosed in U.S. Pat. No.5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in itsentirety. The incorporation of 2′-SR monomer synthons are disclosed byHamm et al., J. Org. Chem., 1997, 62, 3415-3420. 2′-NR₂ nucleosides aredisclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; andPolushin et al., Tetrahedron Lett., 1996, 37, 3227-3230.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O includeS, CH₂, CHF, and CF₂, see, e.g., Secrist, et al., Abstract 21, Program &Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides andtheir Biological Applications, Park City, Utah, September 16-20, 1992,hereby incorporated by reference in its entirety. Additionalmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

As used herein, the term “alkyl” includes but is not limited to straightchain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups ofthe present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50, hereby incorporated by reference in its entirety. As usedherein, the term “lower alkyl” is intended to mean alkyl having 6 orfewer carbons.

As used herein, the term “ara_kyl” denotes alkyl groups which bear arylgroups, for example, benzyl groups. The term “alkaryl” denotes arylgroups which bear alkyl groups, for example, methylphenyl groups. Asused herein the term “aryl” denotes aromatic cyclic groups including butnot limited to phenyl, naphthyl, anthracyl, phenanthryl, and pyrenyl.

As used herein, the term “alkanoyl” has its accustomed meaning as agroup of formula —C(═O)-alkyl. A preferred alkanoyl group is the acetylgroup.

In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocycloalkyl” denotes an alkyl ring system having one or moreheteroatoms (i.e., non-carbon atoms). Preferred heterocycloalkyl groupsinclude, for example, morpholino groups. As used herein, the term“heterocycloalkenyl” denotes a ring system having one or more doublebonds, and one or more heteroatoms. Preferred heterocycloalkenyl groupsinclude, for example, pyrrolidino groups.

In some embodiments of the invention, A is a monocyclic or bicyclicaromatic ring system. Suitable monocyclic or bicyclic aromatic ringsystems include phenyl, naphthyl, pyridyl, furyl and indolyl.

In some preferred embodiments of the compounds and methods of theinvention, R₁₁ and R₁₂ can be, together with the carbon atoms to whichthey are attached, an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms. Examples of such rings include phenyl andnaphthyl. Examples of substituents for such rings include halogen,hydroxyl, alkyl, and acetyl groups. In more preferred embodiments, R₁₁and R₁₂ are each H.

In some preferred embodiments of the invention R₂, or R₃ can be a linkerconnected to a solid support. Solid supports are substrates which arecapable of serving as the solid phase in solid phase syntheticmethodologies, such as those described in Caruthers U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069. Linkers are known inthe art as short molecules which serve to connect a solid support tofunctional groups (e.g., hydroxyl groups) of initial synthon moleculesin solid phase synthetic techniques. Suitable linkers are disclosed in,for example, Oligonucleotides And Analogues A Practical Approach,Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1, pages 1-23.

Solid supports according to the invention include those generally knownin the art to be suitable for use in solid phase methodologies,including, for example, controlled pore glass (CPG), oxalyl-controlledpore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19,1527, hereby incorporated by reference in its entirety), TentaGelSupport—an aminopolyethyleneglycol derivatized support (see, e.g.,Wright, et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporatedby reference in its entirety) and Poros—a copolymer ofpolystyrene/divinylbenzene.

In some preferred embodiments of the invention R₂, R₃ or R_(3a) can be ahydroxyl protecting group. A wide variety of hydroxyl protecting groupscan be employed in the methods of the invention. Preferably, theprotecting group is stable under basic conditions but can be removedunder acidic conditions. In general, protecting groups render chemicalfunctionalities inert to specific reaction conditions, and can beappended to and removed from such functionalities in a molecule withoutsubstantially damaging the remainder of the molecule. Representativehydroxyl protecting groups are disclosed by Beaucage, et al.,Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, ProtectiveGroups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, NewYork, 1991, each of which are hereby incorporated by reference in theirentirety.

Preferred protecting groups used for R₂, R₃ and R_(3a) includedimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl(Pixyl)and 9-(p-methoxyphenyl)xanthen-9-yl(Mox). The R₂ or R₃ group can beremoved from oligomeric compounds of the invention by techniques wellknown in the art to form the free hydroxyl. For example, dimethoxytritylprotecting groups can be removed by protic acids such as formic acid,dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid orwith Lewis acids such as for example zinc bromide. See for example,Greene and Wuts, supra.

In some preferred embodiments of the invention amino groups are appendedto alkyl or to other groups such as, for example, to 2′-alkoxy groups.Such amino groups are also commonly present in naturally occurring andnon-naturally occurring nucleobases. It is generally preferred thatthese amino groups be in protected form during the synthesis ofoligomeric compounds of the invention. Representative amino protectinggroups suitable for these purposes are discussed in Greene and Wuts,Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley &Sons, New York, 1991. Generally, as used herein, the term “protected”when used in connection with a molecular moiety such as “nucleobase”indicates that the molecular moiety contains one or more functionalitiesprotected by protecting groups.

Sulfurizing agents used during oxidation to form phosphorothioate andphosphorodithioate linkages include Beaucage reagent (see e.g. Iyer, R.P., et.al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R. P.,et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide(see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32,3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et.al.,Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (seee.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30, 6757-6760);Bis(O,O-diisopropoxy phosphinothioyl)disulfids (see Stec et al.,Tetrahedron Lett., 1993, 34, 5317-5320);3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996 24,1602-1607, and Nucleic Acids Research, 1996 24, 3643-3644);Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research, 199523, 4029-4033); sulfur, sulfur in combination with ligands like triaryl,trialkyl, triaralkyl, or trialkaryl phosphines. The foregoing referencesare hereby incorporated by reference in their entirety.

Useful oxidizing agents used to form the phosphodiester orphosphorothioate linkages include iodine/tetrahydrofuran/ water/pyridineor hydrogen peroxide/water or tert-butyl hydroperoxide or any peracidlike m-chloroperbenzoic acid. In the case of sulfurization thereaction-is performed under anhydrous conditions with the exclusion ofair, in particular oxygen whereas in the case of oxidation the reactioncan be performed under aqueous conditions.

Oligonucleotides or oligonucleotide analogs according to the presentinvention hybridizable to a specific target preferably comprise fromabout 5 to about 50 monomer subunits. It is more preferred that suchcompounds comprise from about 10 to about 30 monomer subunits, with 15to 25 monomer subunits being particularly preferrred. When used as“building blocks” in assembling larger oligomeric compounds (i.e., assynthons of Formula II), smaller oligomeric compounds are preferred.Libraries of dimeric, trimeric, or higher order compounds of generalFormula II can be prepared for use as synthons in the methods of theinvention. The use of small sequences synthesized via solution phasechemistries in automated synthesis of larger oligonucleotides enhancesthe coupling efficiency and the purity of the final oligonucloetides.See for example: Miura, K., et al., Chem. Pharm. Bull., 1987, 35,833-836; Kumar, G., and Poonian, M. S., J. Org. Chem., 1984, 49,4905-4912; Bannwarth, W., Helvetica Chimica Acta, 1985, 68, 1907-1913;Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65-77, each ofwhich are hereby incorporated by reference in their entirety.

In one aspect of the invention, the compounds of the invention are usedto modulate RNA or DNA, which code for a protein whose formation oractivity it is desired to modulate. The targeting portion of thecomposition to be employed is, thus, selected to be complementary to thepreselected portion of DNA or RNA, that is to be hybridizable to thatportion.

In some preferred embodiments of the methods of the invention, compoundsof Formula II are prepared by reaction of a protected nucleoside havingFormula V:

and a phosphine compound of Formula VI:

in the presence of an acid. Suitable acids include those known in theart to be useful for coupling of phosphoramidites, including, forexample, tetrazole, substituted tetrazoles, dicyanoimidazole, ordiisopropylammonium tetrazolide.

In some preferreed embodiments, compounds of Formula VI are prepared byreacting an alcohol having the Formula XX:

with phosphorus trichloride, and reacting the resultant product,Cl₂P—X₁—(CH₂)₂—X₅—C₆H₄—OX₄, with at least two equivalents of an aminehaving the formula [(R₆)₂N]₂NH. Each of the R₆ groups can be the same ordifferent, and are preferably alkyl having 1 to about 10 carbon atoms,more preferably 1 to 6 carbon atoms, with 3 carbon atoms, andparticularly isopropyl groups, being especially preferred.

In further preferred embodiments, compounds of Formula II can beprepared by reaction of a protected nucleoside of Formula V with achlorophosphine compund of formula ClP(R₅)₂, where R₅ is preferablyisopropylamino, followed by reaction with a compound of Formula XX inthe presence of an acid, for example 1-H tetrazole, substitutedtetrazoles, or dicyanoimidazole, with 1-H tetrazole being preferred.

In some particularly preferred embodiments of the foregoing methods, X₄is benzoyl, acetyl or levulinyl, A is phenyl with the moiety —OX₄ beingin the ortho or para position thereof, with the ortho position beingmore preferred, or A is naphthalene connected to X₅ at the 1-position,with the moiety —OX₄ being in the 5- or 6-position of the naphthalenering.

In the compounds and methods of the present inventon, X₁ and X₂ can eachindependently be O or S. Thus, compounds having chiral phosphoruslinkages are contemplated by the present invention. See Stec, W. J., andLesnikowski, Z. J., in Methods in Molecular Biology Vol. 20: Protocolsfor Oligonucleotides and Analogs, S. Agrawal, Ed., Humana Press, Totowa,N.J. (1993), at Chapter 14. See also Stec, W. J. et al., Nucleic AcidsResearch, Vol. 19, No. 21, 5883-5888 (1991); and European PatentApplication EP 0 506 242 A1, each of which are hereby incorporated byreference in their entirety.

Also provided in preferred embodiments of the invention are compoundshaving the general Formula VII:

wherein X₁, A, X₄ and X₅ and D are as defined above.

In particularly preferred embodiments, the compounds of the inventionhave the Formula II:

wherein:

X₄, M, X₁, R₁, X₂, R₃, B, n, and R₅ are defined as above. In someespecially preferred embodiments of the compounds of the inventionhaving formula II above, X₄ is benzoyl, acetyl or levulinyl, or a groupof formula —(CH₂—CH₂)_(d)Si (R₉)₃ where d is 0 or 1; A is phenyl havingthe moiety —OX₄ is in the ortho or para position, with the orthoposition being preferred, R₅ is diisopropylamino, and n is 0.

The oligomeric compounds of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with the invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms, including warm-blooded animals, can betreated. Further, each cell of multicellular eukaryotes can be treated,as they include both DNA-RNA transcription and RNA-protein translationas integral parts of their cellular activity. Furthermore, many of theorganelles (e.g., mitochondria and chloroplasts) of eukaryotic cellsalso include transcription and translation mechanisms. Thus, singlecells, cellular populations or organelles can also be included withinthe definition of organisms that can be treated with therapeutic ordiagnostic oligonucleotides.

As will be recognized, the steps of the methods of the present inventionneed not be performed any particular number of times or in anyparticular sequence. Additional objects, advantages, and novel featuresof this invention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are intended to beillustrative and not intended to be limiting.

EXAMPLE Example 1 2-Acetoxyphenoxyethyl Alcohol

2-(2-Hydroxyethoxy)phenol (308 g; 2 mol) was taken up in a 5 L Erlnmeyerflask fitted with mechanical stirrer. Anhydrous acetone (4 L, dried overK₂CO₃), and potassium carbonate powder (290 g; 2.1 mol) were added, andthe mixture was stirred vigourously. Acetic anhydride (207 mL; 2.2 mol)was added from an additional funnel slowly over a period of 1 hour.Stiiring was continued for 3 hours. TLC (CH₂Cl₂/MeOH : 9:1, v/v) showeddisapperence of starting material. The reaction mixture was filtered,solid washed thoroughly with acetone (1 L). The combined fractions wasconcentrated and purified by chromatography eluting with hexane andethyl acetate (0% to 35% EtOAc; v/v). The product was obtained as acolorless viscous oil. Yield 258-264 gms (70-72%)

Example 2 General Method for the Synthesis of Phosphoramidites

A 500 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum was assembled under an argon atmosphere. Allglassware were dried at 120° C. for 1 hour. The flask was charged withbis(diisopropylamino) chlorophosphine (84.6 mmol), Hünigs base(diisopropylethylamine) (105.8 mmol) and anhydrous dichloromethane (250mL). With stirring, DMT-protected deoxyribonucleoside (70.5 mmol) wasadded as a solid over a period of 10 minutes. After 30 minutes, all thevolatiles were removed under vacuum (oil pump) and the residue dissolvedin anhydrous acetonitrile (150 mL). A solution of the2-acetoxyphenoxyethyl alcohol (105.7 mmol) in acetonitrile (100 mL) wasadded followed by 1H-tetrazole (63 mmol). Stirring was continued for afurther 1 hour. The reaction mixture was then concentrated, and theresidue redissolved in dichloromethane (500 mL), washed with sodiumbicarbonate solution and dried (Na₂SO₄). Concentration of the driedsolution afforded the crude material which was purified by silica gelflash chromatography. The fractions corresponding to the amidites werecombined and concentrated to afford the pure product as a foammy solid.Yields 65-80%.

Example 3 Preparation of 2-acetoxyphenoxyethyl N,N-diisopropylphosphoramidite

A 500 mL three-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon was assembled under argon atmosphere. Allglassware was dried in an oven at 120° C. for 1 hour. The reaction flaskwas charged with anhydrous ether (150 mL) and phosphorous trichloride(9.27 g; 67.5 mmol). 2-Acetoxyphenoxyethyl alcohol (50 mmol) in ether(100 mL) was added to the reaction flask slowly with stirring at 0° C.(ice cooling) using pressure-equalized addition funnel. After additionwas complete, ice bath was removed and the reaction was stirred forthree hours. The reaction mixture then was transferred to a 500 mL flaskand concentrated under reduced pressure. To this product in anhydrousether (200 mL) was added diisopropylamine (57.7 mL) at 0° C. underargon. After the addition was complete, stirring was continued at roomtemperature for 16 hours (overnight). The reaction mixture was filteredand concentrated to afford the product.

Example 4 General Method for the Synthesis of Phosphoramidites

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum was assembled under an argon atmosphere. Allglassware was dried at 120° C. for 1 hour. The flask was charged with5′-O-DMT nucleoside (7 mmol) and 1H-tetrazole (5.6 mmol). Anhydrousacetonitrile (50 mL) was added. To this stirred mixture under argon atroom temperature was added a solution of 2-acetoxyphenoxyethylN,N-diispropylphosphoramidite (10.5 mmol) in acetonitrile (50 mL). Usualworkup followed by purification afforded the phosphoramidites.

Example 5 Synthesis of T—T Phosphorothioate Dimer

100 milligram (4 mmole) of 5′—O—Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage was taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with dichloromethane and then with acetonitrile.Then, a 0.2 M solution of5′-O-(4,40-dimethoxytrityl)thymidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes. The aqueous solution wasfiltered, concentrated under reduced pressure to give phosphorothioatedimer of T—T.

Example 6 Synthesis of C-T Phosphorothioate Dimer

100 milligram (4 mmole) of 5′—O—Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage was taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution ofN⁴-Benzoyl-5′—O—(4,40-dimethoxytrityl)-2′-deoxycytidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes and then incubated at 55° C.for 12 hours. The aqueous solution was filtered, concentrated underreduced pressure and then treated at room temperature with 1.0 Msolution of tetra-n-butyl ammonium fluoride in THF to give aphosphorothioate dimer of dC-T.

Example 7 Synthesis of 5′-TTTTTTT-3′ Phosphorothioate Heptamer

50 milligram (2 mmole) of 5′—O—Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5′-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2 M solution of5′—O—(4,4′-dimethoxytrityl)thymidine-3′—O—(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05 M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5′-hydroxyl group. The product is washed withacetonitrile.

This complete cycle is repeated five more times to get the completelyprotected thymidine heptamer. The carrier containing the compound istreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature. The aqueous solution is filtered, concentrated underreduced pressure to give a phosphorothioate heptamer of TTTTTTT.

Example 8 Synthesis of 5′-d(GACTT)-3′ Phosphorothioate Tetramer

50 milligram (2 mmole) of 5′—O—Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage was taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution of5′—O—(4,4′-dimethoxytrityl)thymidine-3′—O—(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile wasadded, and reacted at room temperature for 5 minutes. The product waswashed with acetonitrile, and then a 0.05 M solution of Beaucage reagentin acetonitrile was added and reacted at room temperature for 5 minutes.This sulfurization step was repeated one more time for 5 minutes. Thesupport was washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF was added tocap the unreacted 5′-hydroxyl group. The product was washed withacetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) wasadded to deprotect the 5′-hydroxyl group. The product was washed withacetonitrile. Then, a 0.2 M solution ofN⁴-benzoyl-5′—O—(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′—O—(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) wasadded to deprotect the 5′-hydroxyl group. The product was washed withacetonitrile. Then, a 0.2 M solution ofN⁶-benzoyl-5′—O—(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′—O—(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in anhydrous acetonitrile and a 0.4 M solution of 1H-tetrazole inacetonitrile was added, and reacted at room temperature for 5 minutes.The product was washed with acetonitrile, and then a 0.05 M solution ofBeaucage reagent in acetonitrile was added and reacted at roomtemperature for 5 minutes. This sulfurization step was repeated one moretime for 5 minutes. The support was washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added to cap the unreacted 5′-hydroxyl group. Theproduct-was washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) wasadded to deprotect the 5′-hydroxyl group. The product was washed withacetonitrile. Then, a 0.2 M solution ofN²-isobutyryl-5′—O—(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′—O—(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile were added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes at room temperature and thenincubated at 55° C. for 16 hours. The aqueous solution was filtered, andconcentrated under reduced pressure to give a phosphorothioate tetramerof 5′-d (GACTT) -3′.

Example 9 Synthesis of Fully-modified5′-d(TCC-CGC-CTG-TGA-CAT-GCA-TT)-3′ Phosphorothioate 20-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 10 Synthesis of Fully-modified5′-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3′ Phosphorothioate 20-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 11 Synthesis of Fully-modified5′-d(GCG-TTT-GCT-CTT-CTT-CTT-GCG)-3′ Phosphorothioate 21-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 12 Synthesis of Fully-modified5′-d(GTT-CTC-GCT-GGT-GAG-TTT-CA)-3′ Phosphorothioate 20-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid in toluene(volume/volume). Sulfurization was performed using a 0.2 M solution ofphenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for 2minutes. At the end of synthesis, the support was washed withacetonitrile, cleaved, deprotected and purified in the usual manner.

Example 13 Synthesis of Fully-modified5′-d(TCC-CGC-CTG-TGA-CAT-GCA-TT)-3′ Phosphorothioate 20-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 14 Synthesis of Fully-modified5′-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3′ Phosphorothioate 20-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 15 Synthesis of fully-Modified5′-d(GCG-TTT-GCT-CTT-CTT-CTT-GCG)-3′ Phosphorothioate 21-mer

The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 16 Acetylation of 2-(2-hydroxyethoxy)phenol

A 4 liter flask equipped with a mechanical stirrer, and an additionalfunnel is assembled under an atmosphere of argon. All the glassware isdried at 120° C. for 1 h. 2-(2-Hydroxyethoxyphenol) (508.8 g, 3.3 mole)was added to the flask as a solid and dissolved in anhydrous methylenechloride (2.2 L). Triethylamine (1.38 L, 9.9 mole) was added slowlyfollowed by the addition of acetic anhydride (934 mL; 9.9 mole) at roomtemperature slowly over a period of 2 h. The reaction mixture becomesslightly warm. The reaction mixture was stirred at room temperatureovernight. The reaction mixture was diluted with methylene chloride (1L), washed with a solution of saturated sodium bicarbonate (tilleffervescence is complete), brine, dried over (MgSO₄) and concentrated.The crude material was distilled to afford 716 g (91%, b.p. 133-135°C./0.1 mm) of the title compound as a colorless viscous liquid.

Example 17 Chemoselective Hydrolysis using Bisacetate of2-(2-hydroxyethoxy)phenol

A 1 liter flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum was assembled under an atmosphere of argon. All theglassware was dried at 120° C. for 1 h. The bisacetate of2-(2-hydroxyethoxyphenol) (35 g) was added to the flask and dissolved inanhydrous tetra-hydrofuran (350 mL). n-Butanol (52.5 mL) was added tothe mixture followed by the addition of PLY Lipase (type II, Sigma). Thereaction mixture was stirred at room temperature for 48 h (HPLCmonitoring). The mixture was then filtered, the solid was washed withethyl acetate (400 ml) and the combined fractions concentrated.Purification of the material by flash chromatography gave the 28 g ofthe title compound as a colorless viscous liquid.

Example 18 Hydrolysis of the Bisacetate of 2-(2-hydroxyethoxy)phenolusing recycled enzyme

A 1 liter flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum was assembled under an atmosphere of argon. All theglassware was dried at 120° C. for 1 h. The bisacetate of2-(2-hydroxyethoxyphenol) (35 g) was added to the flask and dissolved inanhydrous tetrahydrofuran (350 mL). n-Butanol (52.5 mL) was added to themixture followed by the addition of PPL Lipase (type II, Sigma). Thereaction mixture was stirred at room temperature for 48 h (HPLCmonitoring). The mixture was then filtered, the solid washed with ethylacetate (600 mL) and the combined fractions concentrated. Purificationof the material by flash column chromatography gave 26.8 g of the titlecompound as a colorless viscous liquid.

Example 19 Synthesis of 2Õ-(2-hydroxyethoxy)acetophenone

A 1 liter flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2Õ-Hydroxyacetophenone (13.6 g)is added to the flask and dissolved in xylene (350 mL). Ethylenecarbonate (17.6 g) is added to the mixture followed by the addtion ofsolid powdered potassium carbonate (55.2 g). The reaction mixture isstirred and refluxed for 12-15 h, cooled, filtered, and concentrated.Purification of the material by flash chromatography affords the titlecompound.

Example 20 Baeyer-Villiger oxidation of 2Õ-(2-hydroxyethoxy)acetophenone

A 250 mL flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2Õ-(2-Hydroxyethoxy)acetophenone(1.79 g, 10 mmol) is added to the flask and dissolved in anhydrousmethylene chloride (25 mL). To this stirred solution at room temperatureis added m-chloroperbenzoic acid (50-60% or any technical grade) as asolid. The reaction mixture is cooled to 5° C. and trifluoroacetic acidis added dropwise over a period of 5 min. The reaction mixture isprotected from light and allowed to warm to room temperature. After 5 h,the reaction mixture is diluted with methylene chloride (25 ml),filtered, and the filtrate washed with a solution of saturated sodiumcarbonate, brine and dried. Concentration and purification using flashcolumn chromatography affords the title compound.

Example 21 Synthesis of 4Õ-(2-hydroxyethoxy)acetophenone

A 1 L flask equipped with a magnetic stirrer, a gas inlet for argon, anda septum is assembled under an atmosphere of argon. All the glassware isdried at 120° C. for 1 h. 4Õ-(2-Acetoxyethoxy)acetophenone (22.22 g) isadded to the flask and dissolved in methanol (150 mL). Powderedpotassium cyanide (13 g) is added to the solution and stirred at roomtemperature for 3 h. The reaction mixture is concentrated to a solid,taken up in minimum amount of methylene chloride and passed through apad of silica gel. Concentration of the eluate affords the titlecompound.

Example 22 Baeyer-Villiger oxidation of 4Õ-(2-hydroxyethoxy)acetophenone

A 250 mL flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 4Õ-(2-Hydroxyethoxy)acetophenone(1.79 g, 10 mmol) is added to the flask, dissolved in anhydrousmethylene chloride (25 ml). To this stirred solution at room temperatureis added m-chloroperbenzoic acid (50-60% or any technical grade) as asolid. The reaction mixture is cooled to 5° C. and trifluoroacetic acidis added dropwise over a period of 5 min. The reaction mixture isprotected from light and allowed to warm to room temperature. After 5 h,the reaction mixture is diluted with methylene chloride (25 mL),filtered, and the filtrate washed with saturated sodium carbonatesolution, brine and dried. Concentration and purification using flashchromatography affords the title compound.

Example 23 Synthesis of Fully Protected Diol

A 250 mL flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2-(2-Hydroxyethoxy)phenol (7.71g) is added to the flask and dissolved in anhydrous methylene chloride(100 mL). Ethyl vinyl ether (3.97 g) is added to the solution followedby the addition of catalytic amount of PPTS. The reaction mixture isstirred at room temperature for 3 h. Triethylamine is added followed bythe addition of acetic anhydride. The mixture is stirred at roomtemperature for 6 h, concentrated, taken up in ethyl acetate (150 mL),washed with a solution of sodium bicarbonate, brine, dried andconcentrated. The crude title compound is used as such in the subsequentreaction.

Example 24 Hydrolysis of ethoxyethyl ether

A 250 mL flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. The fully protected2-(2-hydroxyethoxy)phenol is taken up in methylene chloride andn-propanol is added to it followed by the addition of catalytic amountof pyridinium tosylate at 5° C. After stirring the mixture for 12 h,work up and purification by flash column chromatography affords thetitle compound.

Example 25 Synthesis of bis tert-butyldimethylsilyl ether of2-(2-hydroxyethoxy)phenol

A 250 mL flask equipped with a magnetic stirrer, a gas inlet for argon,and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2-(2-Hydroxyethoxy)phenol (7.71g; 0.05 mole) is added to the flask and dissolved in anhydrous methylenechloride (100 mL). Triethylamine (20.2 g, 0.2 mole) is added to thesolution followed by the addition of tert-butyldimethylsilyl chloride(18.09 g; 0.12 mole). A catalytic amount of DMAP is added to acceleratethe reaction. The reaction mixture is stirred at room temperature for 12h and then worked up and purified by flash column chromatography to givethe title compound.

Example 26 Selective hydrolysis of bis tert-butyldimethylsilyl ether of2-(2-hydroxyethoxy)phenol

Bis tert-butyldimethylsilyl ether of 2-(2-hydroxy-ethoxy)phenol (0.01mole) is taken up in methanol. Then 1 wt % (10 mg/mL) of solid iodine isadded and the reaction monitored by tlc. Upon consumption of thealcoholic silyl ether, solid sodium metabisulfite is added and stirreduntil iodine color disappears. The methanolic solution is diluted withmethylene chloride (120 mL), washed with saturated sodium bicarbonate,brine and dried. Purification by flash column chromatography affords thetitle compound.

It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A compound having the formula:

wherein A is a diradical derived from a monocyclic or bicyclic aromaticring system; R₁₁ and R₁₂ are each H, independently alkyl, aryl,heteroalkyl, heteroaryl, alkaryl, or aralkyl; or R₁₁ and R₁₂ togetherwith the carbon atoms to which they are attached form an optionallysubstituted aliphatic or aromatic ring having from 4 to 6 ring atoms; X₄is alkaryl, aralkyl, sulfonyl, thiol, substituted sulfonyl, orsubstituted thiol, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S; R₅ is—N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ring containingfrom 4 to 7 atoms, and having up to 3 heteroatoms selected fromnitrogen, sulfur, and oxygen or optionally one of said R₅ groups ischloro; and R₆ is straight or branched chain alkyl having from 1 to 10carbons.
 2. The compound of claim 1 wherein A is phenylenyl with themoiety —OX₄ in the ortho or para position, or A is naphthalidenylconnected to X₅ at the 1-position, with the moiety —OX₄ being in the 5-or 6-position, and X₁ and X₅ are each O.
 3. The compound of claim 2wherein X₄ is benzoyl, acetyl, or levulinyl.
 4. The compound of claim 3wherein one of said R₅ groups is —N(R₆)₂ where R₆ is isopropyl.
 5. Thecompound of claim 4 wherein one of said R₅ groups is chloro.